Hello everybody,
I am doing library prep for paired end BISULFITE seq on Illumina GAII. I am a little bit confused about the amount of DNA that is required for sequencing. They say 8 pM. After adaptor ligation and size selection I have fragments around 400 bp.
I found this formula to convert from pM to ng:
ug = (pmol * N * 660)/ 10^6
where:
pmol=8
N=400
but this corresponds to 2.1 MICROGRAMS of DNA!
After size selection of the 400 bp fraction I performed bisulfite treatment and then PCR. I should have a geometric increase of my DNA after each cycle, but my DNA is (must be) damaged from the harsh conditions of the bisulfite reaction, because I don't get as much as expected after PCR.
That means I need to go up with the number of PCR cycles (which I don't want), but even then I'm not sure I can reach 2 ug!
Am I wrong in my calculation? Any experience with the same type of experiment?
Thanx a lot!
I am doing library prep for paired end BISULFITE seq on Illumina GAII. I am a little bit confused about the amount of DNA that is required for sequencing. They say 8 pM. After adaptor ligation and size selection I have fragments around 400 bp.
I found this formula to convert from pM to ng:
ug = (pmol * N * 660)/ 10^6
where:
pmol=8
N=400
but this corresponds to 2.1 MICROGRAMS of DNA!
After size selection of the 400 bp fraction I performed bisulfite treatment and then PCR. I should have a geometric increase of my DNA after each cycle, but my DNA is (must be) damaged from the harsh conditions of the bisulfite reaction, because I don't get as much as expected after PCR.
That means I need to go up with the number of PCR cycles (which I don't want), but even then I'm not sure I can reach 2 ug!
Am I wrong in my calculation? Any experience with the same type of experiment?
Thanx a lot!
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